The Art of Muscle Biopsy in the New Genetic Era: A Narrative Review.

Muscle biopsy has been practiced as a well-established part of paraclinical workup in patients with neuromuscular diseases since late 1960s. In this narrative review paper, the role of some pre and post muscle biopsy factors and their importance in achieving the best diagnostic results will be explained. Considering the new advances in diagnostic molecular techniques and the availability of local standard myopathology laboratory as well as the presence of a dedicated myopathologist, the indications of muscle biopsy in four major types of neuromuscular diseases will be shortly reviewed. Moreover, indications of muscle biopsy in four major types of neuromuscular diseases will be shortly discussed based on literature review of recent published diagnostic algorithms and our 11 years' experience of performing about 4000 muscle biopsies cases in the only standard referral diagnostic center for muscle biopsy in Iran. Although diagnostic algorithms of some muscle diseases have been changed based on recent advances in biochemical and molecular diagnostic techniques, still muscle biopsy continues to play a major role in the diagnosis and management of variety of neuromuscular disorders and has proved to be a preferable diagnostic procedure by some neuromuscular specialists for the cases who can benefit from rapid therapeutic management. The application of diagnostic algorithms should be practiced in accordance with geographic distribution of the diseases, the availability of diagnostic techniques and the presence of specialists in each center considering the local insurance coverage and the cost to be paid by the patient in every center.


Introduction
Muscle biopsy has been considered as an essential part of paraclinical workup of patients with myopathic and neuropathic (Neurogenic) diseases alongside with physical examination, laboratory testing, electromyography and molecular investigations.
The credit of morphologic study of muscle biopsy is historically given to the French Neurologist Guillaume-Benjamin-Amand Duchenne (1).
Generally based on histologic findings of muscle biopsy the physician can determine if a muscle is normal or abnormal but even a normal muscle biopsy does not exclude neuromuscular diseases.
Histopathologic findings of a muscle biopsy could be either specific for diagnosis or nonspecific.
Therefore, all histopathological findings should be interpreted in the light of each patient's phenotype.

Case 2
A 34-year-old woman presented to emergency room with loss of conciseness, generalized muscle weakness and severe respiratory distress. She had profound metabolic acidosis and went under ventilator. Her biochemical assays showed normal results. The diagnosis of "lipid storage myopathy" was reported to the physician on the same day of muscle biopsy ( Figure   2). Treatment with a cocktail of high dose Riboflavin, Carnitine and Q10 supplement was started for her. She showed dramatic response and the muscle power of all tested muscles were normalized within one month follow up.

Case 1
A 5-year-old boy adopted from unknown parents presenting with difficulty of rising from floor and climbing stairs for a couple of months with no skin lesion. His serum CK reported to be about 2000. EMG study revealed myopathic pattern. MLPA study of Dystrophin gene presented no mutation. Then WES was requested in which no pathogenic mutation was found. Finally, the little adopted boy who was going to lose his new family by clinical impression of progressive hereditary muscle disease underwent muscle biopsy.
The diagnosis of "juvenile dermatomyositis sine dermatitis" was obtained by muscle biopsy (Figure 1).
Immunosuppressive therapy started for him right after biopsy result. In his next 2-months follow up he had normal physical examination. the daily life activities of the patient after biopsy.
We need to make sure that the selected muscle has not been recently subjected to electromyography (EMG) needling or a previous biopsy or injury.
In cases with selective muscle involvement when the affected muscles are completely gone and the remaining muscles are spared, choosing the right site for muscle biopsy is a big challenge.
Eventually, biopsy of the end-stage muscles should be avoided. There is no established turnaround time for the final muscle biopsy pathology report along with histochemical and immunohistochemical stains (8). In our clinical practice based on the first frozen H&E slide findings a short hand writing diagnostic report will be communicated to our colleagues in cases when immediate therapeutic intervention is required.

Muscle biopsy in four major categories of muscle diseases
In what follows, based on recent published diagnostic algorithms and our 11 years' experience of performing about 4000 muscle biopsies cases in the only standard referral diagnostic center for muscle biopsy in Iran, the indications of muscle biopsy in four major types of neuromuscular diseases will be shortly reviewed. As it is outside of the scope of this narrative review article to go through all the details the tips for the most important and common diagnoses will be briefly mentioned.

Muscle biopsy in metabolic myopathies
Metabolic myopathies are a heterogeneous group of genetic disorders that may be present at any

Case 2
A 34-year-old woman presented to emergency room with loss of conciseness, generalized muscle weakness and severe respiratory distress. She had profound metabolic acidosis and went under ventilator. Her biochemical assays showed normal results. The diagnosis of "lipid storage myopathy" was reported to the physician on the same day of muscle biopsy ( Figure 2). Treatment with a cocktail of high dose Riboflavin, Carnitine and Q10 supplement was started for her. She showed dramatic response and the muscle power of all tested muscles were normalized within one month follow up.
We also perform muscle biopsy at the same time results used to be essential guidance for genetic studies of muscular dystrophies but presently it is mostly recommended for the confirmation of the rhabdomyolysis. In these cases, muscle biopsy is also important for definite diagnosis (9)(10)(11).
Muscle biopsy remains the gold standard for diagnosis of mitochondrial diseases, especially for mtDNA diseases, because of heteroplasmy and tissue-specific mutations (12).

Muscle biopsy in inflammatory myopathies
Inflammatory LGMDs revealed that beta Sarcoglycanopathy is the most common type of sarcoglycanopathy in Iran (24). We recommend to start with a muscle biopsy in suspected cases of sarcoglycanopathies and then the diagnosis of sarcoglycanopathy can be confirmed by MLPA study of sarcoglycan genes. Accordingly, a non-expensive definite diagnosis can be provided for most cases of sarcoglycanopathy in Iran.

Muscle biopsy in structural myopathies
Congenital myopathies are a clinically, molecularly and morphologically a heterogeneous group of muscle diseases and are mainly due to defects in sarcomeric proteins. Unlike its label the presentation of symptoms could be presented later in adulthood.

Discussion
As it was nicely proposed in a book review of the first edition of "Pathology of Skeletal Muscle" "Muscle biopsy is an art" (30). When a muscle biopsy is requested, we must make sure that the highest quality is achieved to provide the correct diagnostic information. There are many pre and post important muscle biopsy factors to be considered in order to achieve the best diagnostic results from a muscle biopsy and multidisciplinary meetings will guarantee the correct interpretation of histologic findings to achieve the best results to the benefit of the patient. Although a major limiting factor of muscle biopsy is its invasiveness still it is not considered as an absolute barrier to request muscle biopsy, because it is minimally Of course the shortcomings remain the author's.